Hindawi Publishing Corporation Journal of Amino Acids Volume 2013, Article ID 979016, 15 pages http://dx.doi.org/10.1155/2013/979016

Review Article Urotensin-II Ligands: An Overview from Peptide to Nonpeptide Structures

Francesco Merlino,1 Salvatore Di Maro,1 Ali Munaim Yousif,1 Michele Caraglia,2 and Paolo Grieco1

1 Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy 2 Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, 80138 Naples, Italy

Correspondence should be addressed to Paolo Grieco; [email protected]

Received 2 December 2012; Accepted 14 January 2013

Academic Editor: Giuseppe De Rosa

Copyright © 2013 Francesco Merlino et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Urotensin-II was originally isolated from the goby urophysis in the 1960s as a vasoactive peptide with a prominent role in cardiovascular homeostasis. The identification of human isoform of urotensin-II and its specific UT receptor by Ames et al. in1999 led to investigating the putative role of the interaction U-II/UT receptor in multiple pathophysiological effects in humans. Since urotensin-II is widely expressed in several peripheral tissues including cardiovascular system, the design and development of novel urotensin-II analogues can improve knowledge about structure-activity relationships (SAR). In particular, since the modulation of the U-II system offers a great potential for therapeutic strategies related to the treatment of several diseases, like cardiovascular diseases, the research of selective and potent ligands at UT receptor is more fascinating. In this paper, we review the developments of peptide and nonpeptide U-II structures so far developed in order to contribute also to a more rational and detectable design and synthesis of new molecules with high affinity at the UT receptor.

1. Introduction species, the cyclic hexapeptide sequence, c[Cys-Phe-Trp- Lys-Tyr-Cys], is conserved in all isoforms (Figure 1,orange Urotensin-II (U-II) belongs to a series of regulatory neu- residues). ropeptides first isolated from the urophysis of the teleost fish The length of urotensin-II peptides is variable across Gillichthys mirabilis by Karl Lederis and Howard Bern in the species and it ranged from 17 amino acid residues in mice 1960s. This cyclic peptide derived from goby fish, H-Ala- to 11 in humans, depending on the proteolytic cleavages Gly-Thr-Ala-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH, was that occur in precursors. The N-terminus region of U-II originally characterized on the basis of its interesting smooth is highly variable among animal species [5], whereas the muscle contracting and hypertensive effects. It has been long C-terminal amino acids, organized in a disulphide-linked considered that U-II was exclusively produced by the fish cyclic array, c[Cys-Phe-Trp-Lys-Tyr-Cys], are continuously urophysis [1].However,theidentificationofU-IIfromthe conserved from species to species, suggesting their primary brain of a frog [2, 3] has shown that the cDNA encoding role in the biological activity [6]. In addition, the goby prepro-U-II exists in several species of vertebrates. Moreover, isoform of U-II exhibits some structural similarities with thegeneisexpressednotonlyinthecaudalportionofthe somatostatin-14 concerning especially the presence of a spinal cord but also in brain neurones, from frogs to humans disulphide-linked cyclic core at their C-terminus portion [4]. In fact, urotensin-II isopeptides are present in several containing the biologically active domain Phe-Trp-Lys [7]. species of vertebrates, and although the amino acid sequence Moreover, the characterization of cDNA encoding carp pro- in the N-terminus of urotensin-II peptides diverges across U-II has shown that the U-II and somatostatin-14 precursors 2 Journal of Amino Acids share a common structure organization since the active altered since pathogenesis of several cardiovascular disorders peptides are both located at the C-terminal portion of the provokes an upregulation of UT receptor and of U-II resulting precursors [8]. Later, once the UT receptor was identified as a in vasoconstriction. member of family, some somatostatin- To date, findings in molecules with high affinity tothe like peptides containing a disulphide bridge, such as human urotensin-II system has led to discovering new ligands, melanin-concentrating hormone (MCH), somatostatin-14, peptide, and nonpeptide analogues. This review is aimed at cortistatin-14, and octreotide, were screened on UT receptor investigating the structure-activity relationship on urotensin- in order to compare the resulting biological activities with II system by analyzing peptide and nonpeptide structures so that of the endogenous U-II [9]. By these observations it was far developed in order to contribute also to a more rational established that U-II represented the only endogenous and detectable design and synthesis of new molecules with with high affinity for the somatostatin-like receptor named high affinity at the UT receptor. UT receptor. The human UT-II (hU-II) is a cyclic undecapeptide, H- Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH, rec- 2. SAR Studies on U-II ognized as the natural ligand of an orphan G-protein coupled receptor, first named as a rat receptor with high affinity The potential therapeutic application of urotensin-II system for U-II, GPR14 [10–12]. Subsequently, a human G-protein has continuously stimulated structure-activity relationship coupled receptor showing 75% similarity to the orphan rat studies (SARs), which could elucidate the structural features receptor was cloned and renamed UT receptor by IUPHAR of this important hormone. On the other hand, the discovery [13]. The role of UT receptor has been continuously inves- of new analogues acting as agonist or antagonist is extremely tigated and nowadays it is commonly accepted that it is important to explore the physiological role of hU-II. First widely distributed in the CNS and in different peripheral structural studies performed on U-II by nuclear magnetic tissues including cardiovascular system [14], kidney, blad- resonance (NMR) spectroscopy [23–25] have revealed that der, prostate, and adrenal gland [10, 15–17]. This exten- the peptide adopted a preferential conformation both in sive expression has revealed the multiple pathophysiological DMSO and water solution, which did not show any classical effects mediated by the hUT/UT receptor interaction such as secondary structures. Amino acids in the core region of U- II are identified in a highly compact conformation with the cardiovascular disorders (heart failure, cardiac remodelling, 4 7 8 and atherosclerosis), smooth muscle cell proliferation, renal formation of a hydrophobic pocket, in which Ala ,Phe ,Trp , 12 disease, diabetes, and tumour growth [18]. Nevertheless, UT and Val are projected on the same side of the molecule receptor is especially expressed in vascular smooth muscle, being at least in part important residues for the binding site. endothelium,andmyocardiumandplaysakeyroleinthe In contrast, hU-II and some analogues resulted to be fold in regulation of the cardiovascular homeostasis. Furthermore, defined secondary structure when dissolved in SDS solution, this receptor has much structural homology to members of a membrane mimetic environment [26]. the somatostatin and receptor family and it could be activated Subsequently, with the aim of investigating the role played by somatostatin-14 and cortistatin at micromolar doses [10]. by the exocyclic region in the receptor interaction, a series The coding for the UT receptor has been located in of truncated peptides related to hU-II has been investigated. 17q25.3 [19]. Truncation studies are of prime importance for the detection The hU-II binds with high affinity to this receptor, result- of the minimal sequence of peptide required to retain the bio- ing in intracellular calcium mobilization via phospholipase logical activity. The effect of sequential deletion of exocyclic C-dependent increase in inositol phosphates. In isolated rat residues from N- or C-termini in hU-II sequence does not thoracic aorta fragments hU-II induces contraction mediated appear to be significant in the calcium-mobilizing potency by two distinct tonic and phasic components. However, its and efficacy, whereas the removal of any residue that belongs vasoconstrictor activity is well observed in primate arteries, to the cyclic region determines reduction or total absence of in which it causes a concentration-dependent contraction thebiologicalactivity.Theshortestandfullypotentsequence of isolated arterial rings with an EC50 valuelessthan1nM, of U-II was individuated in the octapeptide U-II(4–11),H- meaning a 10-fold more potency than endothelin-1. However, Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]Val-OH, that preserves the the in vivo effects can depend on the species, type of potency at the human UT receptor, showing similarity to blood vessel, concentration of U-II, route of administration, somatostatin-14 in which truncation of the segment led to and results of tissue and species in exam, and they can active analogues. be also contradictory. Accordingly, the peptide also elicits The importance of a free amino group in the N-terminal vasodilatory effects on the small arteries of rats and on the of the resulted octapeptide U-II(4–11) was evaluated by its resistance arteries of humans, probably due to the release modification in the succinoyl derivative performed in 2002 by of endothelium-derived hyperpolarizing factor and nitric Coy et al. [27]. The peptide was extremely potent according to oxide [20].Inahealthyhuman,U-IIbehavesasachronic data from the biological activity, EC50 = 0.12 ± 0.03 nM, and regulatorofvasculartoneratherthaninfluencingtissuesin binding affinity, 𝐾𝑖 = 1.14±0.01 nM and 𝐾𝑖 = 1.65±0.04 nM, aphasicmanner[21]. U-II binds to its receptor in a “pseudo- at human and rat UT receptor, respectively. Additional 4 irreversible” manner, and slow dissociation rate from the UT dicarboxylic residues introduced in substitution of Asp have receptor leads to prolonged activation of the receptor and a showed similar results. Furthermore, the replacement of this functionally silent system [22]. This state of homeostasis is amino acid with the corresponding amidated Asn gives a Journal of Amino Acids 3

Human U-II S S

1 2 3 4 5 6 7 8 9 10 11 H2N Glu Thr Pro Asp Cys Phe Trp Lys Tyr Cys Val COOH

Goby U-II S S

1 2 3 4 5 6 7 8 9 10 11 12 H2N Ala Gly Thr Ala Asp Cys Phe Trp Lys Tyr Cys Val COOH

Frog U-II S S

1 2 3 4 5 6 7 8 9 10 11 12 13 H2N Ala Glu Asn Leu Ser Glu Cys Phe Trp Lys Tyr Cys Val COOH

Rat U-II S S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 H2N Gln His Gly Thr Ala Pro Glu Cys Phe Trp Lys Tyr Cys Val COOH

Figure 1: A comparison of urotensin-II (U-II) isopeptides sequences isolated from different species of vertebrates.

Table 1 Name Peptide sequence

PRL-2903 H-4Fpa-c[Cys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH2

SB-710411 H-Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-NH2 󸀠 󸀠 BIM-23127 H-D(2 )Nal-c[Cys-Tyr-DTrp-Orn-Val-Cys]-(2 )Nal-NH2 󸀠 BIM-23042 H-DNal-c[Cys-Tyr-DTrp-Lys-Val-Cys]-(2 )Nal-NH2 8 [Orn ]U-II H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Orn-Tyr-Cys]-Val-OH P5U H-Asp-c[Pen-Phe-Trp-Orn-Tyr-Cys]-Val-OH Urantide H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH UFP-803 H-Asp-c[Pen-Phe-DTrp-Dab-Tyr-Cys]-Val-OH URP H-Ala-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH Urocontrin H-Bip-c[Cys-Bip-Trp-Lys-Tyr-Cys]-Val-OH

GSK248451 H-Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-NH2 compound that retains a full activity in all three assay systems, as conotoxins, endothelin-1, and somatostatin analogue that suggesting that in this position an amino acid negatively gave interesting results by the same modification29 [ ]. Start- charged in the side chain is not required. However, the ingfromtheminimumactivefragmentU-II(4–11),introduc- 4 Nle -analogue lacks of potency showing that the –CH2COX tionofthelactambridgeinanappropriatelengthledtopep- carbonyl group present in both the Asp and Asn side chains tides that maintain bioactivity to the detriment of potency, isimportantprobablybecauseofitspossibilitytoactasan suggesting that the size of the lactam bridge is a crucial acceptor for hydrogen bond with the UT receptor. The side parameter. Peptide analogues synthesized in this study were chain can also contain an aromatic ring substituted with polar characterized by ring-closing sequence that ranges from 20 groups such as OH and NO2, which is of great interest in the to 24 atoms and, interestingly, the smallest peptide sequence, development of antagonists based on the previously identified having the same length as the native peptide containing the somatostatin antagonist octapeptides. disulphide bridge, does not show any biological activity. In The cyclic structure is essential for hU-II and McMaster contrast, peptide analogue characterized by a larger ring, et al. in 1986 [28] reported a lack of biological activity for containingOrnandAspasresiduesinthe22atomslactam the corresponding “ring-opened” analogue. In 2002 Grieco bridge, behaved as a full agonist, but was approximately 100- et al. considered the replacement of the disulphide bridge by fold less potent than hU-II. Thus, replacement of the Cys-Cys a side-chain-to-side-chain lactam bridge in accordance with cyclic motif could be well tolerated by an appropriate longer observations on several biologically relevant peptides, such lactam bridge despite the partial loss of activity, probably 4 Journal of Amino Acids due to the different orientation of the key amino acid side ledtodramaticdecreaseoftheagonistactivity,suggesting chains. However, in a later work performed by Foister et al. the importance of the side chains of these amino acids and in 2006 [30] a cyclic “cysteine-free” hexapeptide derivative of their spatial orientation for interaction with the UT receptor; 9 U-II, in which Tyr was replaced with a 𝛽-naphthylalanine surprisingly, stereoinversion of L-Trp in D-Trp does not show residue, [Ala-Phe-Trp-Lys-(2)Nal-Ala], bounds the human asignificantchangeoftheEC50 value versus the endogenous 8 UT receptor with higher affinity𝐾 ( 𝑖 = 2.8 nM) than ligand.TheroleofLys was also investigated by replacing it the corresponding disulphide-bridged truncated hexapeptide with lipophilic amino acids and hydrophilic nonbasic amino 𝐾 =95 U-II(5–10) ( 𝑖 nM). Furthermore, modifications of acids that produce inactive peptides [33]. Thus, positive 5 10 the Cys –Cys disulphide bridge, such as the macrocyclic charge represented by primary aliphatic amine of Lys in lactam and the penicillamine-derived disulphide moiety, position 8 is essential for the biological activity. However, couldchemicallystabilizeandrestricttheconformational reducing the distance of the primary aliphatic amine from the peptide backbone led to a progressive reduction of both flexibility of the biologically active cyclic hexapeptide core 8 sequence. In particular, penicillamine residue in replacement potency and efficacy. [Orn ]U-II analogue showed a weak 5 of Cys resulted to be very useful in order to obtain a potent contraction of rat aorta strips corresponding to about 20% 5 agonist, [Pen ]hU-II(4–11), subsequently renamed P5U [29]. of the U-II maximal effect at micromolar concentrations. As potent agonist with reduced conformational flexibility, In position 9 the –OH group of Tyrosine was proved to Lavecchia et al. performed subsequent NMR study in DMSO be replaced with –OCH3,–NO2,–CH3, –F, –H, and –NH2 8 onthispeptidein2005[31]. The positions of Lys amino obtaining any improvement in potency or efficacy, except for 9 group and Tyr aromaticsidechainwereinproximitywith 3-iodo-Tyr residue that produced a full UT receptor agonist, 7 8 adistanceof6.2AandTrp˚ indole and Lys amine were 6-fold more potent than the natural peptide. separated by 5.6 A.˚ Docking of P5U into a hUT homology A further and useful attempt to alter potency and efficacy model based on the structure of bovine revealed versus UT receptor was represented by the introduction of 8 interesting points of interaction. The Lys interacts primarily nonnatural amino acids into the sequence [32]. Replacement 130 100 305 with Asp ,aswellaswithresiduesofTyr and Tyr ; oftheTyrresiduewiththebulkier2-Nal[(2-naphthyl)-L- 9 212 alanine]inthegobyU-IIsequenceshowedsimilarpotency Tyr is accommodated in a binding pocket defined by Lys , 296 281 277 in agonist activity (with a value of EC50 =0.34±0.1nM Val ,Ala ,andTrp particularly involved in a 𝜋-stacking 7 than the value for the goby U-II, EC50 = 0.17 ± 0.05 nM) interaction, and the indole NH of Trp binds to the carbonyl 298 in the functional assay and a 6-fold improvement in affinity of Tyr . in the binding assay (𝐾𝑖 = 0.04 ± 0.02 nM), presumably In the structure-function study performed by Kinney due to enhanced hydrophobic interactions in the tyrosine- et al. in 2002 [32], an alanine scan of truncated goby U- binding pocket. In contrast, the Bip residue, [(2-biphenyl)- II demonstrated that the replacement of Trp, Lys, and Tyr L-alanine] did not show equal result, confirming that larger is crucial for the maintenance of biological activity. The groups are not well accommodated. In this work, Kinney et sequence Trp-Lys-Tyr within U-II is essential for binding and al.alsoprovidedforanovelagonistpharmacophoremodel activation of the receptor, indicating that the hydrophobic through the docking of goby UT-II into a rat UT receptor 7 9 8 side chains of Trp and Tyr and the positive charge of homology model. According to this modelling study, the Lys 8 Lys represent pharmacophoric elements. Moreover, NMR amino group of the ligand was essential for the interaction 130 studies performed by Flohr et al. in 2002 [24]revealed with Asp on transmembrane helix TM3 of UT receptor, that the distances between the pharmacophoric points are with the binding cavity drawn by the extracellular loops that key elements in the development of SAR. Accordingly, first strengthened the accommodation of the ligand. The distances NMR studies applied to the receptor-unbound hU-II in water characterized for the pharmacophoric sequence were 11.2 A˚ 8 9 were developed in order to provide for a putative agonist between the Lys amino and Tyr aryl groups, 8.4 A˚ between 8 7 8 7 pharmacophore. Structural model showed the Lys amino theindoleTrp and Lys amine, and 8.2 A˚ between the Trp 9 9 group and Tyr aryl ring being in proximity with a distance and Tyr aryl groups. 7 9 of 6.4 A;˚ the Trp and Tyr were separated by 12.2 A,˚ and 7 8 Trp aromatic residue and Lys amino group were separated by 11.3 A.˚ Later, another agonist pharmacophore model was performed by studying conformation adopted by a less potent 3. Peptide Ligands analogue without Val residue, that is, Ac-[Cys-Phe-DTrp- As mentioned above, the human U-II is involved in several Lys-Tyr-Cys]-NH2 (200-fold less potent than hUT-II). Since pathophysiological pathways of disorders especially regard- this peptide was more closely similar to the receptor-bound ing cardiovascular system along with the observation that conformation, this structural model suggested new distances the interaction U-II/UT receptor regulates the contractility between pharmacophoric elements of Trp-Lys-Tyr sequence 8 9 7 and growth properties of cardiac and peripheral vascular (Lys amino group and Tyr aryl ring at 11.1 A,˚ Trp and 9 7 vessels led to identifying selective ligands. In particular, Tyr aryl residues separated by 8.3 A,˚ and Trp aryl and 8 since the modulation of the U-II system offers a great Lys amino groups separated by 13.7 A).˚ Flohr et al. also potential for therapeutic strategies related to the treatment of studied the substitution of amino acids in the hexacyclic part cardiovascular diseases, the research of selective compounds of hU-II sequence with the corresponding D isomers. This is more intriguing. Journal of Amino Acids 5

Based on the search for a definitive pathophysiological NH2 role for U-II and its receptor in the cardiovascular home- HN ostasis and aetiology of relating disorders, the design of suitable tool compounds of peptide or nonpeptide nature could be of significant utility. New molecules may assist in determining this role by the development of selective UT receptor antagonists. N HN Therefore, first attempts came out from observations on somatostatin system, due to sharing sequence peculiarities O between hU-II and somatostatin. Indeed, some somato- statin analogues such as PRL-2903 Table 1, H-4Fpa-c[Cys- Pal-DTrp-Lys-Tle-Cys]-Nal-NH2,resultedintheabilityto Figure 2: Structure of S7616. block the hU-II-induced rat aorta ring tone at micromolar concentrations, although it showed low species selectivity [34]. Another peptide somatostatin analogue, described by Coyetal.in2000,showedmoderateaffinityforUTreceptor. in HEK293 cell lines expressing either the human or rat UT This peptide, named SB-710411, H-Cpa-c[DCys-Pal-DTrp- receptor was evaluated by generating hU-II concentration- response curves. These were indeed shifted progressively Lys-Val-Cys]-Cpa-NH2, was able to inhibit U-II-induced contraction in rat isolated thoracic aorta in a surmount- to the right in a parallel manner with no changes in the maximum response to hU-II, suggesting competitive able manner (𝑝𝐾𝑏 = 6.28)[35]. Exposure to 10 𝜇MSB- 710411 causes a significant shift in the agonist concentration- antagonism.Moreover,BIM-23127showedabout0.5log unit lower affinity in competition binding experiments to response curve with no suppression of the 𝐸max,suggesting that it acts as a competitive antagonist. In contrast, SB- human or rat UT receptors and inhibition of hU-II-promoted 710411 did not alter the contractile efficacy of angiotensin-II, intracellular calcium mobilization producing a significant phenylephrine, or KCl, although it potentiated the contractile suppression of the maximum contractile response to hU- response to endothelin-1 in the isolated rat aorta. However, II. In contrast of this noncompetitive antagonism of con- sincelittlewasknownaboutthepharmacologyofthisligand traction to U-II in isolated rat aorta, BIM-23127 inhibited inotherspecies,Behmetal.in2004[36]reportedtheirobser- calcium mobilization in human embryonic kidney 293 cells vations on the pharmacological effects at the rat and monkey expressing UT receptors in a competitive manner. A related receptor antagonist, BIM-23042, H-DNal- recombinant UT receptor because of the weak homology 󸀠 between rodent and primate UT receptors (∼76% of homol- c[Cys-Tyr-DTrp-Lys-Val-Cys]-(2 )Nal-NH2,displayeddiffer- ogy than the homology between monkey and human UT ent functional activities at several UT receptor orthologues. It receptor that are about 97% identical) [37]. SB-710411 acts as a behaved as a full agonist at human and monkey UT receptor, ligand for both the recombinant rat and monkey UT receptor a partial agonist at mouse UT receptor, and a competitive (30–150 nM affinities, 56- and 87-fold less potent than U- antagonist at rat UT receptor [41]. Among the most potent compounds, Camarda et al. in II, resp.). However, functional behaviour of this peptide at 8 these two UT receptor orthologous differs radically. As was 2002 [38] identified the hU-II derivative [Orn ]U-II, that was characterized in vitro as a novel peptide ligand for the reported previously, SB-710411 itself did not promote inositol 8 phosphate formation but inhibited the agonistic actions of UT receptor. Modification of Lys to the nonnatural amino U-II in the rat aorta [35]. In contrast to the rat, SB-710411 acidOrnwassuggestedbychemicalobservationsthatLys behaved as a full agonist at the recombinant monkey UT belongs to the most important sequence for the biological receptor, inducing the maximal response although EC50 was activity. This synthetic analogue behaved as a full agonist in approximately 100-fold less potent than U-II. Thus, despite the calcium functional assay in HEK293 human and rat UT the antagonistic effects of SB-710411 observed at the rat UT cells, inducing similar maximal effects as U-II. However, the 8 receptor, this peptide acts as a full agonist at the monkey UT potency of [Orn ]U-II at both receptors was 3-fold lower than receptor. These findings suggest that the functional response U-II (𝑝EC50 = 7.93 ± 0.16 and 8.06 ± 0.22,resp.,whereasU- of UT receptor modulators at the rodent UT receptor does not II increased intracellular calcium levels in HEK293 hUT and necessarily predict the functional response at nonrodent UT rUT cells with similar high potencies, 𝑝EC50 = 8.51 ± 0.18 receptors, and the incoherence could result from alterations and 8.54 ± 0.14, resp.). In contrast, different results were in receptor number and/or coupling efficiency as well as obtained in the rat aorta bioassay, in which the compound Camarda et al. in 2002 [38] proposed for a different UT behaved as a competitive antagonist, showing only in highest 8 receptor ligand, [Orn ]hU-II. concentrations (10 𝜇M)aweakresidualagonistactivity(25% As cyclosomatostatin octapeptide analogue that shares compared to the maximal effect of U-II). The variance structural similarities with SB-710411, the peptide neu- between results obtained between the cell and tissue assay 󸀠 8 romedin B receptor antagonist BIM-23127, H-D(2 )Nal- could be interpreted assuming that [Orn ]hU-II is a partial 󸀠 c[Cys-Tyr-DTrp-Orn-Val-Cys]-(2 )Nal-NH2 [39], was inves- agonist. tigated by functional activity at recombinant and native UT In 2002 Grieco et al. [29] generated a novel peptide UT receptors [40]. The effect of increasing concentration of BIM- receptor agonist by introduction of an unusual amino acid 23127 on hU-II-induced intracellular calcium mobilization inthedisulphidebridgeofhUT-II(4–11),thepotentanalogue 6 Journal of Amino Acids

O O

O Optimization O

N N

Cl Cl AC-7954 FL68

Figure 3: Structures of AC-7954 and its optimized derivative FL68.

O O

NH NH N Optimization N

Cl

Cl

FL-104 Compound 1

Figure 4: Structures of FL-104 and its optimized derivative compound 1.

OH as the most important for full agonist activity, maintains O the same spatial orientation as in the native peptide. Thus, the chemical modification brought by the unusual more N HN N constrained penicillamine residue mainly influences the H 6 proximal Phe position, leaving Trp, Lys, and Tyr residue nearly unaffected. The enhanced pharmacological properties observedinthecaseofP5Ucanbeassignedtothisconforma- N tional restriction revealing the importance of the exploration of specific orientations in the three-dimensional space by Figure 5: Structure of palosuran (ACT-058362). which amino acid side chains can interact with the receptor. 8 Since antagonist peptides such as SB-710411, [Orn ]U- II, BIM-23127 so far described showed weak potency at UT 5 𝛽 𝛽 receptors with concomitant antagonist activities to different [Pen ]hU-II(4–11),alsoknownasP5U.This , -dimethyl- substituted cysteine residue led to higher conformational receptortypesandbehavedaspartialagonistactivityatUT, rigidity in the sequence of hU-II C-terminal octapeptide. new attempts were challenged in order to develop more Data showed that P5U has a 3-fold higher affinity for the potent and selective UT receptor antagonist. Patacchini et al. in 2003 [42] described the pharmacological activities of UT receptor than the endogenous ligand as competition 5 8 5 7 experiments witnessed its ability to displace the iodinated two compounds: [Pen ,Orn]hU-II(4–11) and [Pen ,DTrp, 8 radioligand with comparable affinity (iodinated hU-II bound Orn ]hU-II(4–11), named urantide (urotensin-II antagonist 𝑝𝐾 = the human UT receptor saturably with high affinity, 𝐷 peptide). Both peptides derived from the hU-II(4–11) frag- 9.2±0.14, whereas P5U has similar affinity to the unmodified ment, previously reported as the minimal active sequence 𝑝𝐾 = 9.7 ± 0.07 5 C-terminal octapeptide hU-II(4–11), 𝑖 ). of hU-II, as well as further replacement of Cys by penicil- In functional experiments on the rat aorta, P5U was 20- lamine, 𝛽,𝛽-dimethylcysteine, were achieved in order to give fold more potent than hU-II and 10-fold more potent than them conformational rigidity stabilizing the putative bioac- hU-II(4–11), being by far the most potent U-II analogue tive conformation. In functional experiments both peptides in the rat thoracic aorta bioassay. Interestingly, conforma- showed no agonist effect by cumulative administration in the 1 tional analysis by [ H] nuclear magnetic resonance (NMR) range 0.1 nM to 10 𝜇M. However, urantide was totally ineffec- spectroscopy combined with molecular modelling on this tive as an agonist even when administered as a single con- 5 8 peptide also indicated further details about structure-activity centration,thatwasnotshownfor[Pen,Orn]hU-II(4–11), relationships since the putative pharmacophoric Trp-Lys-Tyr suggesting a sort of desensitization known to affect UT sequence into the cyclic portion of this analogue, retained receptor-mediated responses in this preparation. As the most Journal of Amino Acids 7

CF3 NH2 H Br N S O O N N N Piperidine N

O 4-Aminoquinoline R HN N Pyrrolidine H O O OCH3 N S O N N N N N H H 4-Ureidoquinoline Piperazine OCH3

N OCH3 2-Aminoquinoline

1,2,3,4-Tetrahydroisoquinole I H H H O O N N N N Cl S N O H Br O Quinolone Diarylsulfonamide

O NH2 N H F NH NH

N NH2 H N N O N F N O H

Biphenylcarboxamide Benzazepine

Figure 6: Structures of nonpeptide urotensin-II receptor antagonist reported in the patent literature.

Cl in both BIM-23127 and SB-710411. Urantide represented an Cl O O extremely potent UT receptor antagonist since it was about S N 50-to100-foldmorepotentthananyothercompounds N O H tested in the rat isolated aorta. Despite of the potent UT receptor antagonist activity in the rat aorta bioassay, urantide Cl F3C showedresidualagonistactivityathumanrecombinantassay in a calcium mobilization assay [44]. In order to develop a selective antagonist, chemical modifications led to generating Figure 7: Structure of SB-611812. 5 7 8 the peptide [Pen ,DTrp,Dab]U-II(4–11),alsoknownas UFP-803, closely related to the urantide sequence[45]. In the present molecule, the residual agonist activity is less than that potent UT receptor antagonist compound so far reported in of urantide. In the rat aorta bioassay, UFP-803 competitively the rat isolated aorta, urantide has also high affinity for the antagonizes U-II contractile action behaving as a selective UT human (𝑝𝐾𝑖 = 8.3)andfortherat(𝑝𝐾𝑖 = 8.3) UT receptors. receptor antagonist. Conformational studies on urantide performed in 2005 by In 2003 a report from Sugo et al. [46] demonstrated 7 Grieco et al. [43] showed that the distance between Trp and the existence of a paralogue of U-II named U-II related 9 Tyr side chains was 11.5 A,˚ greater than that observed in peptide (URP), a novel peptide first isolated from the extract 7 peptide agonist P5U (6.1 A)˚ because of the inversion of L-Trp of rat brain and subsequently also proposed as endogenous into the corresponding D isomer in urantide. The feature of ligand for UT receptor in the rat, mouse, and possibly in inversion of the configuration of the Trp residue in position human. The amino acid sequence was determined as H- 7 was suggested by the presence of the same modification Ala-c[Cys-Phe-Trp-Lys-Tys-Cys]-Val-OH and it exhibits high 8 Journal of Amino Acids

R have showed antagonist properties in rat isolated aorta and O O N partial agonist action by mobilization of intracellular calcium H3CO S in specific recombinant UT receptor HEK/CHO cell systems. N O H Similar observations upon this residual agonist activity have already been made by Kenakin in 2002 [51]andCamarda H3CO Br et al. in 2002 [38] that have proposed an “assay-dependent” R agonism/antagonism resulted from different UT receptor expression and/or signal transduction-coupling efficiency, for SB-706375 CF3 example, depending on the receptor density and the efficiency SB-657510 Cl of receptor couplings. For this reason identification of a novel and selective antagonist was achieved by examining Figure 8: Structure of SB-706375 and SB-657510. ligand-evoked UT receptor agonism under conditions of both low and high receptor density and efficient coupling and amplification.In2006Behmetal.[52] described GSK248451, O N H-Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-NH2 [53, 54], as Cl a potent UT receptor antagonist in all native mammalian N O N H isolated tissues retaining an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5 fold less Cl than observed for urantide). Furthermore, since GSK248451 Figure 9: Structure of SB-436811. represents a selective UT receptor antagonist by blocking the systemic vasopressor actions of exogenous U-II it became a suitable tool compound for further investigations con- cerning the role of U-II in the aetiology of mammalian binding affinity for human UT receptor in transfected cell cardiometabolic diseases. lines and high contractile potency in the rat aortic ring assay, suggesting that some physiological effects could be not completely attributed to U-II. In order to evaluate the correct orientation of amino acid side chains belonging to the cyclic 4. Nonpeptide Ligands region of URP in the activity of the peptide, each amino The use of peptides as drugs in a therapeutic approach acid has been replaced with the corresponding stereoisomer is often problematic because of their poor oral and tissue in a D-scan analysis [47]. D-isomer substitution within 3 5 6 absorption, and their low stability due to the rapid proteolytic the cyclic portion in replacement of Phe ,Lys,andTyr cleavage by enzymes. The pharmacokinetic limits of pep- reduced binding affinity and contractile activity, confirming tides can be generally overcome by developing nonpeptide the primary role of this portion in receptor recognition. In 4 molecules, inspired to the main sequence of the peptide contrast, the [DTrp ]URP analogue retained important bind- andinparticularmimickingthespecificsecondarystructure ing affinity, suggesting relative tolerance in the interaction responsible for the biological activity. Regarding to the design with the receptor by stereoinversion occurring in that posi- 4 of nonpeptide ligands, the conformation and the size of the tion. [DTrp ]URP showed also reduced efficacy appearing to peptidebackboneisoftendifficulttomimicbyusingas behave as a partial agonist with moderate potency and a full scaffold organic molecules and modifications onto hydropho- antagonist with low potency, indicating that point substitu- bic, steric, and electronic properties could generate potential tion of the Trp residue in U-II and URP sequence could lead active compounds and optimize their affinity and selectivity. to the development of antagonists. Therefore, Chatenet et al. Nonpeptide agonists and antagonists at the human UT in 2012 [48] provided the replacement of the indole moiety in receptor could be important tool compounds in determining URP in order to obtain promising antagonists. In particular, the role of U-II and its derivatives in the urotensin system, the introduction of the more hydrophobic uncoded amino 4 andtheyhavebeendevelopedinseveralstudies.Specifically, acid Bip led to the novel antagonist urocontrin, [Bip ]URP. the design and synthesis of selective receptor antagonists The main feature of this peptide was the ability to reduce the should be helpful to clarify the role of human U-II as a efficacy of hU-II but not URP-induced vasoconstriction in multifunctional peptide in mammalian pathophysiological a rat aorta assay. Despite the structural homology between functions. Additionally, a pure nonpeptide antagonist stable U-II and URP and their concurrent expression in several on in vivo administration could be very helpful to provide human tissues, recent studies have reported different actions an alternative pharmacological strategy in different disease for these two peptides such as cell proliferation [49]and models. distinctive myocardial contractile activities [50]. Therefore, Historically, first approaches for the discovery of new the identification of more selective ligands should be helpful nonpeptide ligands at GPCR receptors were based on high- for the rational design of more selective molecules in order to throughput screening (HTS) studies or knowledge of the clarify the role of U-II and URP in the urotensinergic system. 3D structure and secondary conformation adopted by the Several peptide UT receptor antagonists such as urantide, natural ligand. A virtual screening based on 3D pharma- 8 [Orn ]U-II, UFP-803, BIM-23042, and SB-710411 exhibit cophores defined from the key residues of U-II was even contradictory actions in selected assay systems since they performed on an Aventis compounds database by Flohr et Journal of Amino Acids 9

Cl O Cl O N N N Optimization N O of the lead compound O N O O NH

N

N O O Compound 2 Compound 3

Figure 10: Structures of the lead compound 2 and its analogue compound 3 (piperidine derivatives). al. in 2002 [24] in order to identify functional antagonists ofthebicyclicisochromane-basedringsystem.Theproduct of U-II. The screening was based on their two agonist was synthesized as racemic mixture (𝑝EC50 =6.5)that was pharmacophore models: one associated with the human resolved in order to test enantiomers in R-SAT. Interestingly, U-II peptide and one associated with Ac-[Cys-Phe-DTrp- the assay revealed that the isomer (+)-AC-7954 is more potent Lys-Tyr-Cys]-NH2.From500compoundsthatmatchedthe as UT receptor agonist (𝑝EC50 =6.6), indicating that this U-II pharmacophore, the most notable compound was activity is highly stereoselective. The mode of interaction in S7616, 1-(3-carbamimidoyl-benzyl)-4-methyl-1H-indole- of (+)-AC-7954 with human UT receptor was clarified by 2-carboxylic acid (naphthalene-1-ylmethyl)amide, revealing later docking studies [31]. (+)-AC-7954 binds the human UT an IC50 of 400 nM (Figure 2). receptor through interaction between the basic amino group 130 The phenyl ring of the indole and the naphthalenemethy- and Asp locatedonTM3ofthereceptoraswellasthep- lamine side chain are localized onto the two aromatic features chlorophenyl ring located in a hydrophobic pocket, where 9 of the pharmacophore. The basic benzamidine group in S6716 usually Tyr residuebinds,andthebenzoringinteractswith 130 118 was shown to form a charged interaction with Asp residue Phe by an aromatic stacking interaction. within TM3 of the human UT receptor. Here, the basic amino As first nonpeptide agonist the compound named AC- group was considered as crucial feature for all following 7954 has been considered as a lead compound and a series designed antagonists. ofanaloguesofthiscompoundhavebeendevelopedin Because of the absence of specific small molecule UT order to obtain more potent nonpeptide ligands at the receptor agonists, Croston et al. performed a functional human UT receptor [56]. In accordance with this study mammalian cell-based R-SAT assay for high-throughput the isochromanone core has been kept intact, whereas new screening in 2002 [55]. In this assay the UT receptor was bulkier amino groups and introduction of substituents in multiplexed with vectors for the expression of additional position 4 or in the aromatic rings were investigated so receptor targets, such as the muscarinic M3 receptor and that the structure-activity relationship study around AC- some orphan GPCRs, in order to increase the number of 7954 could complete the knowledge about U-II/UT receptor drug-target interactions tested without altering the response interaction. These several structural modifications led to both and sensitivity characteristics of potential ligands. Screening increased and decreased activities, although beneficial effects a library of 180000 small diverse organic molecules tested in a were obtained when substituents were introduced in the multiplexed R-SAT assay, AC-7954, 3-(4-chlorophenyl)-3-(2- aromatic part of the isochromanone ring system, whereas (dimethylamino)ethyl)isochroman-1-one, was identified as a more sterically demanding amino groups resulted to be novel nonpeptide agonist with a potency of 300 nM at the damaging to the activity. The 6,7-dimethyl derivative of AC- human UT receptor (Figure 3). 7954 showed the most potency among the series (𝑝EC50 = This compound is selective to activate the UT receptor, 6.87 ± 0.03) and once its racemate has been resolved into although the other receptors included in the multiplex the pure enantiomers, it was indicated the (+)-enantiomer, screening assay belong to somatostatin and subsequently named FL-68 (Figure 3), as the own potential classes that are the closest to UT receptor for genetic sequence active stereoisomer (𝑝EC50 = 7.30).Fromtheseriesof homology. Having no significant response when tested at compounds synthesized in this study, FL-68 was found the concentrations up to 15 𝜇M on several receptor targets, most interesting since it was very active versus UT receptor including dopamine (D1, D2, D5), muscarinic (M1, M3, M5), and not showing at the same time any activity versus the serotonin (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5- closely related somatostatin receptors. HT2B, 5-HT2C), histamine (H2), 𝛽-adrenergic (𝛽-1, 𝛽-2), Although the isochromanone-based agonists so far somatostatin (sst-2, sst-3, sst-5), CRF-1, CRF-2a, CRF-2b, described were interesting for their druglikeness properties 𝜅-opioid, adrenomedullin, and CCK-a receptors, AC-7954 and their high selectivity for the UT receptor, in 2007 resulted to be a highly selective nonpeptide agonist of the UT Lehmann et al. [57]chosetoobtainnewmoleculesbybreak- receptor. This compound has low molecular weight, drug- ing of C3–C4 bond in the isochromanone scaffold. This syn- like lipophilicity, a basic amino function (𝑝𝐾𝑎 = 8.7),and thetic strategy adopting for development of novel urotensin- it exhibits limited conformation flexibility for the presence II agonists was primarily focused to the introduction of 10 Journal of Amino Acids

F H3C H R N F N N OCH3 OCH3 Optimization OCH3 N of the lead compounds N O OCH3 O OCH3 N O OCH3 N N N H

O O O NH S O S O

Compound 4 Compound 5 (JNJ-28318706) Compound 6 (JNJ-39319202)

Figure 11: Structures of piperazino-isoindolinone derivatives compound 4 and compound 5 (JNJ-28318706) were optimized into the novel compound 6 (JNJ-39319202).

𝑝 7.64 ± 0.23 HN agonist compound with a EC50 value of ,which is higher than the precursor FL-104. N As pharmacological tool in determining physiological and pathological roles of endogenous U-II in kidney diseases, N H in 2004 Clozel et al. [59] reported the discovery and charac- terization of a specific and potent inhibitor of the human UT Figure 12: Structure of compound 7. receptor, the compound palosuran (ACT-058362) (Figure 5). Results from radioligand binding experiments carried out in membrane preparations from CHO cells expressing the human UT receptor indicated palosuran as potent inhibitor 125 of I-U-II, binding at human UT receptor with an IC50 different linkers between the two aromatic rings. Thus, a 125 series of ether, ester, amide, sulfonamide, carbamate, and urea of 3.6 ± 0.2 nM, whereas radioligand I-U-II was potently derivatives were performed. These more flexible compounds inhibitedbytheunlabeledU-IIwithanIC50 value of 1.2 ± led to molecules most retained in activity and efficacy com- 0.2 nM.OncelllinessuchasTE-671andCHO,theinhibitory pared to AC-7954 except for ethers and sulfonamide probably binding potency of palosuran toward the human UT receptor duetotheabsenceofconformationaleffectsinducedbythe was lower than on membranes (IC50 = 46.2 ± 13 nM pharmacophoric carbonyl group. Interestingly, esters showed on TE-671 cells and IC50 =86±30nM on recombinant lower efficacy than other derivatives, whereas the introduc- CHO cells). Binding studies showed also that the inhibitory tion of larger and lipophilic substituents in the variable binding potency of palosuran is higher more than 100- aromaticpartofthemoleculetendgenerallytoincrease fold on human UT receptor compared with the rat UT potency. To investigate the improvement of the efficacy it receptor (IC50 = 3.6 nM toward the human receptor, seems that the introduction of electron-donating groups con- IC50 valueof1475nMforratreceptor).However,palosuran tributed positively. Furthermore, among benzamide series inhibited U-II-induced concentration of rat aortic rings in more lipophilic compounds were approximately 1 order of a potent and concentration-dependent manner and inhib- magnitude more potent than the other amides. Accordingly, ited the maximal contractile response of the rings almost −4 the biphenylamide derivative, later known as FL-104, was completely at 10 M suggesting an insurmountable kind of recognized as a potent agonist (𝑝EC50 = 7.49 ± 0.03)andits antagonism of contraction induced by U-II. Despite this, racemic mixture was resolved in order to evaluate the main in binding studies using membrane preparations expressing active stereoisomer, identified in (+)-FL-104 isomer (𝑝EC50 = human UT receptor, palosuran interacted competitively with 7.49), considerably more active than (−)-FL-104. The (+)- its receptor. This controversial mode of inhibition observed S-enantiomer of FL-104 was considered as one of the most between functional assays and receptor binding studies could potent nonpeptide agonists known (Figure 4). be linked to the likely noncompetitive antagonism due to Analogues of FL-104 were designed and synthesized in the partial internalization of the UT receptor after binding order to enable comparisons between SAR in the isochro- with palosuran. This could reduce the receptor availability manone and benzamide series of UT agonists [58]. By on the cell surface in the isolated rat aortic ring system. the evaluation of the distance between the aromatic rings Functional assays showed that palosuran was a selective and the dimethylamino group and by the replacement of antagonist of UT receptor not antagonizing the action of the dimethylamino group with a piperidine moiety new other vasoconstrictor agents such as KCl, endothelin-1, 5- compounds were obtained and tested for their ability to hydroxytryptamine, and norepinephrine. Therefore, palo- stimulatethehumanUTreceptorinanR-SATassay.How- suran represents an important tool of nonpeptide nature in ever, compound 1 was the most interesting among the series ordertovalidatetheroleofendogenousU-IIindisease (Figure 4). Its racemate mixture was resolved and the (+)- models,inparticularinkidneypathologies.Forthisreason, (S)-enantiomer corresponded to the most active nonpeptide palosuran was considered as an interesting tool and a novel Journal of Amino Acids 11 and interesting UT receptor antagonist for evaluating the U-II antagonist for the human UT receptor (≥100-fold) pathophysiological role of endogenous U-II in renal system, compared to 86 distinct receptors including ion channels, since both U-II and UT receptor are highly expressed in the enzymes, transporters, and nuclear hormones (𝐾𝑖/IC50 > kidney [15]. Endogenous U-II plays a role in mediating the 1𝜇M). The contractile responses induced in isolated aortae abnormal renal vasoconstriction after ischemia and short- by KCl, phenylephrine, angiotensin-II, and endothelin-1 were term intravenous administration of palosuran reduced the unaltered by SB-706375 (at 1 𝜇M concentration). glomerular and tubular dysfunction and renal tissue injury The very closely related compound SB-65751067 [ ]was induced by renal ischemia [59]. alsoindicatedaspotentantagonistinisolatedarteriesfrom Further evidence that the 4-ureido-quinoline core, also rats, cats, monkeys, and hUT-transgenic mice (Figure 8). The shared with the structure of palosuran, could be taken as a characterization of this compound led to the first nonpeptide promising template for antagonists turned out from several UT receptor radiolabel, namely, the tritiated radiotracer 3 patent applications. In addition, several examples of UT [ H]SB-657510 [66]. receptor antagonists have appeared in the patent literature Substituted diarylsulfonamides, reported in other patent but they were less discussed in this paper. Thus, 4-ureido- applications, possessed significant affinity for UT receptors quinoline derivatives, in which 1,2,3,4-tetrahydroisoquinole, (𝐾𝑖 ∼1𝜇M). They were designed as UT receptor antagonists piperidine, piperazine, and pyrrolidine moieties were intro- and CCR-9 antagonists for the treatment of congestive heart duced, were also tested for their ability to displace human 125 failure, stroke, ischemic heart disease, and so forth [68]. [ I]U-II binding to a rhabdomyosarcoma cell line (IC50 Other UT antagonist series was represented by values ranging from 1 to 1000 nM) [60–62]. Other non- aminoalkoxy benzyl pyrrolidine derivatives even reported peptide molecules reported in patent applications were byGlaxoSmithKline.BasedonanHTSprotocolinvolving based on 4-aminoquinolines [63]andquinolone,suchas2- hU-II-mediated calcium mobilization in hUT-expressing aminoquinolines and 2-aminoalkylquinolin-4-ones deriva- HEK293 cells, the lead compound SB-436811 was identified tives [64], template (Figure 6). for its moderate potency (𝐾𝑖 = 200 nM) binding to Researchers at GlaxoSmithKline conducted extensive human UT receptor but weak potency in rat hUT binding biological studies leading to the discovery of a series of aryl- (𝐾𝑖 = 3.2 𝜇M) [69](Figure 9). As group linker between the sulfonamide derivatives developed from high-throughput substituted-phenyl moiety and the heterocyclic ring, the screening. The compound SB-611812 showed potent binding sulfonamide group was replaced with an alkyl group. at the rat UT receptor (𝐾𝑖 = 121 nM) and based on its antagonist activity in rat aortic tissue and interesting Biphenylcarboxamide and benzazepine scaffolds were pharmacokinetic properties such as high bioavailability (∼ also reported in patent applications since these derivatives 100%) and half-life (∼5 h), this compound was proposed demonstrated highly potent UT receptor antagonism [70]. as a useful pharmacological tool (Figure 7). Accordingly, a In particular, most structurally different from other UT coronary artery ligation study was conducted in rats with receptor antagonists, benzazepines represented one of the this lead UT antagonist since patients with congestive heart most potent antagonists at the human UT receptor so far ∼2 failure (CHF) are usually associated with high level of U- described (IC50 nM). II and UT receptor in the heart tissue [65]. In this study, With the aim to identify a compound with low nanomolar treatment with SB-611812 (30 mg/kg per day) for eight weeks potency toward both rat and human UT receptor, as research significantly reduced overall mortality, left ventricular end- tool for evaluating the role of U-II/UT receptor interaction diastolic pressure, lung edema, right ventricular systolic pres- in disease models, Luci et al. in 2007 [71]developednew sure, central venous pressure, cardiomyocyte hypertrophy, series of small organic molecules. New lead structures and ventricular dilatation, underlying the importance of were obtained by executing an HTS protocol involving a hUT-II in this disorder. functional assay based on cells transfected with rat UT The identification and characterization of compound SB- receptor, a fluorometric imaging plate reader (FLIPR) to 706375 was originally described by Douglas et al. in 2005 measure intracellular calcium flux, and the potent peptide 󸀠 [66](Figure 8). This compound was identified as novel UT UT agonist Ac-c[Cys-Phe-Trp-Lys-(2 )Nal-Cys]-NH2 [32]. receptor antagonist, acting in a surmountable, reversible By the application of this assay to a large compound library, manner with high affinity across species (binding 𝐾𝑖 = various 4-phenylpiperidine-benzoxazin-3-ones derivatives 4.7–20.7 nM) and good selectivity. The potent antagonist were identified. Compound 2, which contained a 4-(4- 125 activity was demonstrated by its ability to inhibit [ I]hU- chlorophenyl)piperidine subunit, was identified as moder- II binding to both mammalian recombinant and “native” UT ately potent compound (IC50 =7.1𝜇M). On the basis of receptors with a reversible mode of action (𝐾𝑖 = 4.7 ± 1.5 to this lead compound more analogues were elaborated and 20.7 ± 3.6 nM at rodent, feline, and primate recombinant UT compound 3 (Figure 10) that, containing an aryl-substituted receptors; 𝐾𝑖 = 5.4 ± 0.4 nM at the endogenous UT receptor piperidine subunit, was found to be a more potent antagonist in SJRH30 cells). The antagonist activity was also validated toward both rat and human UT receptor (IC50 =10nM at in a number of assays such as the inhibition of contraction rat UT receptor and 𝐾𝑖 =65nM at human UT receptor). For of the rat isolated aorta (𝑝𝐾𝑏 = 7.47)andtheinhibitionof this reason, this compound was selected for in vivo evaluation intracellular calcium mobilization in HEK293 cells express- and its efficacy was shown in reversing the ear-flush response ing UT receptor (𝑝𝐾𝑏 = 7.29–8.00). SB-706375 was a selective inducedbyU-IIinrats. 12 Journal of Amino Acids

Other classes of compound that turned out from this several disorders and physiological effects. The synthesis screening were represented by piperazino-phtalimide and of U-II analogues has been performed adopting two main piperazino-isoindolinone derivatives, very different struc- strategies based on the design and development of peptide tural type of UT receptor antagonists from those previously or nonpeptide derivatives. Both the approaches present sev- reported in literature. Thus, Lawson and coworkers in 2009 eral advantages and disadvantages. Although peptides offer [72] realized these novel series starting from the identification several advantages as therapeutic tools over small organic of the compound 4 (rat FLIPR EC50 = 0.54 nM, rat UT bind- molecules, when they are composed of natural amino acids ing 𝐾𝑖 = 0.12 nM) and the strictly related compound 5, also they are not very good drug candidates because of their known as JNJ-28318706. The latter had improved metabolic intrinsic physicochemical and pharmacokinetic properties stability and improved potency (rat FLIPR IC50 =84nM), such as low bioavailability and biodistribution. On the other and (R)-enantiomer exhibited also good oral bioavailability hand, as drug candidates in a therapeutic strategy, peptides in pharmacokinetics experiments. However, as piperazino- offer greater efficacy, selectivity and specificity, and lower isoindolinone derivative (Figure 11), the compound 6 (JNJ- toxicity than small organic molecules since they represent 39319202) showed single-digit nanomolar potencies in the thesmallestfunctionalpartofaprotein.However,the rat FLIPR assay (IC50 = 1.0 nM) and in the human UT introduction of pharmacophoric elements in small organic receptor binding assay (𝐾𝑖 = 4.0 nM). This compound also structures is fascinating and favourable in the treatment of exhibited potent antagonism in the human calcium flux assay some diseases. Thereby, organic compounds represent the key (IC50 = 8.0 nM). Moreover, a recent study reported a facile forobtainingpromisingdrugcandidatesbyreachingeasilya alkylation-cyclization reaction performed on isoindolinone compromise between stability and selectivity. core of JNJ-39319202 that yielded to novel tricyclic derivatives The research of the best therapeutic tools in disorders with retained potency in antagonism activity [73]. produced by U-II could be achieved by chemical optimiza- Aminomethylpiperazines derivatives were also drawn out tion strategies for urotensin-II derivatives, peptides or non- from the structure belonging to 𝜅-opioid receptor agonists peptide analogues. The best knowledge of structure-activity [74]. In accordance with this study, optimization of the relationships may provide useful chemical requirements in piperazine moiety provided high affinity urotensin-II recep- order to improve pharmacodynamic and pharmacokinetic tor antagonists (more than 100-fold selectivity over the 𝜅- properties such as bioavailability, reduction of elimination opioid receptor) and, among this series, specific compounds and biodegradation by proteolytic enzymes activity, low inhibited urotensin-induced vasoconstriction in isolated rat toxicity, reduced drug-drug interactions, and short half-life aortic ring. values avoiding accumulation of metabolites in tissues. As Other 2-aminomethyl piperidine derivatives had mod- for urotensin peptides, substitution of natural amino acid erate hUT binding affinity𝐾 ( 𝑖 = 400 nM) and hUT residues by unnatural amino acid such as D-stereoisomer, functional activity (FLIPR IC50 = 600 nM) [75]. However, nonproteogenic constrained amino acid or 𝛽-amino acid these series were correlated to problems such as cytochrome hasrepresentedstrategicchemicalapproachresultinginthe P450 inhibition and low oral bioavailability. Therefore, some increase of stability and/or affinity for the UT receptor. improvements were obtained by using a piperazine core in At least, development of nonpeptide antagonists at the UT such promising compounds. Subsequently, removal of the receptor is an attractive alternative as pharmacological tool piperidine and piperazine linker groups led to a series of in the identification of the role of endogenous U-II in several potent biphenylmethyl derivatives. pathophysiological effects. Wang et al. in 2008 [76] described a series of N-alkyl- 5H-pyrido[4,3-b]-indol-1-amines as UT receptor antagonists References since the tricyclic compound 7 was identified in an HTS as a lead compound due to its binding affinity𝑝𝐾 ( 𝑖 = 8.1)and [1]H.A.Bern,D.Pearson,B.A.Larson,andR.S.Nishioka, submicromolar antagonist activity (𝑝IC50 = 6.3)athuman “Neurohormones from fish tails: the caudal neurosecretory UT receptor (Figure 12). system. I. “Urophysiology” and the caudal neurosecretory system of fishes,” Recent Progress in Hormone Research,vol.41, pp. 533–552, 1985. 5. Conclusion [2] J. M. Conlon, F. O’Harte, D. D. Smith, M. C. Tonon, and H. 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